PROJECT SUMMARY Macrophages (MF) are one of the largest immune cell components of tumor lesions, where their numbers can even exceed tumor cells. Though MF are important for tissue homeostasis and control of infections, in tumors MF adopt a phenotypic state which confers them with pro-tumorigenic and immunosuppressive functions. Because of this, MF are a target for cancer treatment. However, modulating tumor-associated MF has proved difficult. This is in large part because we still do not have a complete understanding of the genes that control many key aspects of MF biology, and thus we still lack an optimal target for modulating relevant MF functions. The objective of this project is to identify genes that drive effector and suppressor immune activity of human MF. In single cell RNA-seq analysis of human non-small cell lung carcinoma (NSCLC), my sponsor labs identified a large module of genes upregulated in tumor-associated MF compared to MF in adjacent healthy lung tissue. This included a number of genes with immunosuppressive functions, but for a majority of the identified genes, the functions, particularly as they may relate to immunity, are not known. Almost half of the identified genes encode for factors with potential upstream regulatory roles in MF biology, such as transcription factors (TF), signaling components, and cell surface receptors. In this project, I will test the hypothesis that the immunosuppressive activity of tumor-associated MF is driven by the regulatory factors and signaling components upregulated on intratumoral MF. To test my hypothesis, I will utilize a novel CRISPR genomics platform my sponsor lab developed, which enables high-dimensional phenotyping of multiple CRISPR knockouts simultaneously and with single cell resolution (Wroblewska, Dhainaut et al. Cell 2018). I will systematically knockout the tumor-associated MF signature genes and determine how perturbation impacts key phenotypes and functions of the cells, including expression of checkpoint ligands and modulation of T cell activation. By identifying genes that control MF identity and effector and suppressor function, the outcome of this project will provide new insights in to the molecular mechanics of one of the major cells controlling the direction of the immune response in the tumor microenvironment and provide molecular targets for therapeutic interventions aimed at enhancing anti-tumor immunity.